The overall goal of the work described in this proposal is to develop a mechanistic understanding of TGN and vacuole membrane protein sorting and assembly in the simple eukaryote Saccharomyces cerevisiae. Yeast has proved to be an excellent model system both for identifying the proteins regulating membrane traffic in eukaryotic cells and for investigating the molecular mechanisms by which these proteins function. The focus of this proposal to investigate the molecular mechanisms of TGN retention and V-ATPase biosynthesis, assembly, transport and targeting. Mutational analysis has revealed that TGN retention of DPAP A involves both static retention and retrieval signals, and the Grdl9p has been found to bind directly to the cytosolic domain of DPAP A. The Grd19p complex will be characterized, and the region of the DPAP A tail to which it binds will be analyzed. Characterization of the grd mutants has revealed that the Na+/H+ exchanger Nhx1p is required for transport out of the late endosomal/prevacuolar compartment. The role for Nhxlp ion transport will be investigated to learn how endosomal membranes sense the lumenal ion environment. There are two isoforms of the V-ATPase in yeast, and the Golgi/endosomal isoform is retained outside the vacuole due to targeting signals in the cytosolic domain of Stvlp, one of two 100 kDa integral membrane subunit isoforms. These signals will be further characterized by deletion and site-directed mutagenesis, and the proteins that function to retain Stv1p outside the vacuole will be identified by genetic and two-hybrid analyses. Previous genetic analysis has identified three genes that encode proteins that function in the assembly of the vacuolar proton-translocating ATPase (V-ATPase) in the yeast endoplasmic reticulum (ER). One of these proteins, Vma21p, appears to escort the V-ATPase complex out of the ER once the V-ATPase is assembled. A combined genetic and biochemical approach will be taken to investigate the potential role of Vma21p as an assembly chaperone, vesicle packaging protein, and an escort for the V-ATPase complex. The other two assembly factors, Vmal2p and Vma22p, will also be investigated for roles in assembly of the complex and for packaging of the V-ATPase into vesicles budding from the ER.